CN109142291B - Method for typing and identifying microorganisms by using fluorescent sensing array - Google Patents
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Abstract
The invention belongs to the field of molecular biological materials, and discloses a fluorescence sensing array and a method for typing and identifying microorganisms by adopting the same. The fluorescence sensing array is formed by mixing an aggregation-induced emission molecule solution and a graphene oxide solution. According to the fluorescence sensing array, aggregation-induced emission molecules are directly mixed with graphene oxide, typing identification of various microorganisms can be achieved, chemical modification is not needed, signal response is completed within 2h, data can be acquired through an enzyme-labeling instrument, a fluorescence spectrometer and digital imaging, and the data is processed by combining principal component analysis, so that the fluorescence sensing array has the advantages of safety, simplicity, convenience, high efficiency, no complex chemical markers, low background signals and strong specificity.
Description
Technical Field
The invention belongs to the field of molecular biological materials, and particularly relates to a fluorescence sensing array and a method for typing and identifying microorganisms by adopting the same.
Background
Bacteria and fungi have close relationship with human beings, on one hand, the number of deaths due to bacterial or fungal infections worldwide reaches 2 million each year; on the other hand, bacteria in the intestinal tract play an important role in food digestion, synthesis of nutrient molecules and immunity of the body. Based on this, typing and identifying microorganisms is of great significance to our understanding and utilization of microorganisms. However, the current microorganisms are mainly based on microbial culture, which has the disadvantages of time-consuming and the like, and some of the microorganisms require a special culture medium for culture. Other techniques, including PCR, gene sequencing, surface enhanced Raman and mass spectrometry, are also complex and time and labor intensive. The development of novel and efficient microorganism typing and identification technology has important significance.
Biological pattern recognition techniques have proven to be a powerful approach to the typing of complex biomolecules. Have been used to distinguish proteins, ions and volatile gases. In general, biological recognition technology is mainly based on the construction of molecular sensing arrays. Specific patterns are generated for different analytes based on non-specific interactions between the analyte and the molecular sensing array. Currently, typing identification of bacteria or tumor cells using this technique has been reported. However, so far, the following problems still exist: 1, complex equipment is often used in signal acquisition, 2, traditional fluorescent molecules often have strong fluorescent signals, and 3, at present, living microorganisms are often used for identification and differentiation of bacteria, and the microorganisms generally have certain infectivity.
Disclosure of Invention
In view of the above disadvantages and shortcomings of the prior art, it is a primary object of the present invention to provide a fluorescence sensing array. The fluorescence sensing array is constructed by fluorescence molecules (AIEgenes) with aggregation-induced emission (AIE) properties and Graphene Oxide (GO). Unlike conventional fluorescent molecules, aggregation-induced emission fluorescent molecules have very weak luminescence in a dissolved state, and their fluorescence emission efficiency is greatly enhanced when molecular motion is restricted. The graphene oxide can further quench background fluorescence and introduce the interaction between competitive biomolecules, so that the distinguishing capability of the molecular sensing array is improved.
The invention also aims to provide a method for typing and identifying microorganisms by using the fluorescence sensing array.
The invention further aims to provide a rapid microorganism typing and identifying kit prepared by adopting the fluorescence sensing array.
The purpose of the invention is realized by the following technical scheme:
a fluorescence sensing array is formed by mixing an aggregation-induced emission molecule solution and a graphene oxide solution; the aggregation-inducing luminescent molecule has a structural formula as described in any one of A1-A13:
(A1,Anal.Chem.2010,82,7035-7043；A2,Chem-Eur.J.2010,16,1232-1245；A3,Chem.Commun.2017,53,4795-4798；A4,Chem.Commun.2012,48,8637-8639；A5,J.Med.Chem.2007,50,663-673；A6,Chem.Sci.2017,8,1822-1830；A7,Chem.Sci.2017,8,1822-1830；A8,Chem-Eur.J.2010,16,1232-1245；A9,Anal.Chem.2015,87,9487-9493；A10,Chem.Commun.2014,50,8312-8315；A11-A13,CN.Patent.105541660[P].2016-01-15)。
preferably, the aggregation-inducing luminescent molecule has a structural formula as described in any one of a1 to a7 as follows:
preferably, the concentration of the aggregation-induced emission molecule solution is 1-10 mM, the concentration of the graphene oxide solution is 0.2-0.4 mg/ml, and the volume ratio of the aggregation-induced emission molecule solution to the graphene oxide solution is 1 (0.5-5).
A method for typing and identifying microorganisms by adopting the fluorescence sensing array comprises the following steps:
and uniformly mixing the fluorescence sensing array and the microbial lysate for incubation, and performing typing identification on the microbes according to the difference of the generated fluorescence signals.
Further, the incubation time was 2 h.
Further, the generated fluorescence signal is collected by a microplate reader or a fluorescence spectrometer, and the collected signal realizes typing identification of the microorganism by a Principal Component Analysis (PCA) method.
Further, the microorganisms include fungi, gram-negative bacteria and gram-positive bacteria, and specifically include candida albicans, saccharomyces cerevisiae, pseudomonas aeruginosa, staphylococcus aureus, escherichia coli and bacillus subtilis.
A microorganism rapid typing identification kit prepared by adopting the fluorescence sensing array.
The principle of the invention is as follows: the fluorescence of the fluorescent molecular array constructed by aggregation-induced emission molecules and graphene oxide is completely quenched before the fluorescent molecular array acts on the microbial lysate, and the microbial lysate is used as an analysis substrate to light the fluorescent molecular array. So that the fluorescence of the fluorescent molecular array can be recovered to different degrees after the incubation with the microbial lysate. The fluorescence sensor array has different fluorescence response intensities to different microbial lysates, so that specific fluorescence patterns are generated for specific microbes, and finally, the differentiation of different microbes is realized by combining a mode recognition technology.
Compared with the prior art, the fluorescence sensing array and the identification method have the advantages and beneficial effects that:
(1) according to the fluorescence sensing array, aggregation-induced emission molecules are directly mixed with graphene oxide, typing identification of various microorganisms can be achieved, chemical modification is not needed, signal response is completed within 2h, data can be acquired through an enzyme-labeling instrument, a fluorescence spectrometer and digital imaging, and the data is processed by combining principal component analysis, so that the fluorescence sensing array has the advantages of safety, simplicity, convenience, high efficiency, no complex chemical markers, low background signals and strong specificity.
(2) The identification method of the present invention selects a lysate of a microorganism as an analysis substrate, which can provide more molecular information about the microorganism than the outer membrane of the microorganism. Meanwhile, the bacterial lysate can be stored for a long time more conveniently.
Drawings
FIG. 1 is a fluorescent photograph of a fluorescence sensor array according to example 1 of the present invention before and after addition of a bacterial lysate (E.coli).
FIG. 2 is a fluorescent photograph of a fluorescent molecular sensor array constructed in example 2 of the present invention after interaction with six microbial lysates. X1-X7 in the abscissa represent seven AIE molecules A1-A7, respectively, and Y1-Y6 in the ordinate represent six microorganisms, respectively.
FIG. 3 shows fluorescence intensity signals extracted after interaction between the fluorescent molecule sensor array and six kinds of microbial lysates in example 2 of the present invention.
FIG. 4 is a signal pattern of six microorganisms obtained by subjecting fluorescence signals generated from lysates of six microorganisms to a principal component analysis in example 2 of the present invention.
Fig. 5 is a fluorescent photograph and a relative fluorescence intensity chart of two sets of fluorescent molecular sensor arrays (a, not containing graphene oxide, b, adding graphene oxide) added with bacterial lysate (pseudomonas aeruginosa and escherichia coli) in example 3 of the present invention.
FIG. 6 is a schematic diagram illustrating the principle of the competitive effect induced by graphene oxide according to the present invention.
Detailed Description
The present invention will be described in further detail with reference to examples and drawings, but the present invention is not limited thereto.
Example 1
The fluorescence sensing array and the method for typing and identifying the microorganisms in the embodiment specifically comprise the following steps:
(1) preparing a solution of AIE molecules; respectively a1 (water soluble, 1mM), a2 (water soluble, 10mM), A3(DMSO soluble, 1mM), a4(DMSO soluble, 1mM), a5(DMSO soluble, 10mM), a6(DMSO soluble, 1mM), a7(DMSO soluble, 1 mM); the AIE molecules A1-A7 have the following structural formula:
(2) and respectively adding 10 mu L of AIE molecular solution into a 96-well plate, wherein the number is 1-7, adding 50 mu L of graphene solution (the concentration is 0.4mg/ml) into a No. 1-6 tube, adding 5 mu L of graphene solution (the concentration is 0.2mg/ml) into a No. 7 tube, and uniformly mixing to prepare the fluorescent molecular sensor array.
(3) And (3) adding 100 mu L of bacterial lysate (escherichia coli) into each test tube in the step (2), uniformly mixing, incubating for 2h at room temperature, and photographing under the irradiation of an ultraviolet lamp.
The photographs of the fluorescence sensing array in this example before and after addition of the bacterial lysate (E.coli) are shown in FIG. 1. It can be seen from fig. 1 that the fluorescence of the fluorescence sensing array is quenched before the addition of the bacterial lysate, and only the fluorescence of the material of the 96-well plate is present, whereas the fluorescence of the fluorescence sensing array is illuminated after the addition of the bacterial lysate, and each well emits fluorescence of a different color and brightness.
Example 2
The fluorescence sensing array and the method for typing and identifying the microorganisms in the embodiment specifically comprise the following steps:
(1) preparing a solution of AIE molecules; respectively a1 (water soluble, 1mM), a2 (water soluble, 10mM), A3(DMSO soluble, 1mM), a4(DMSO soluble, 1mM), a5(DMSO soluble, 10mM), a6(DMSO soluble, 1mM), a7(DMSO soluble, 1 mM);
(2) respectively adding 10 mu L of AIE molecular solution into a 96-well plate, wherein the number of the AIE molecular solution is X1-X7, adding 50 mu L of graphene solution (the concentration is 0.4mg/ml) into a tube No. X1-X6, adding 5 mu L of graphene solution (the concentration is 0.2mg/ml) into a tube No. X7, and uniformly mixing to prepare the fluorescent molecular sensor array.
(3) Six groups of samples are loaded in parallel, and fluorescence sensing arrays for six microorganisms are prepared and are numbered as Y1-Y6.
(4) Six kinds of bacterial lysate (100. mu.L) were added to each well. Y1-Y6 correspond to Candida albicans, Saccharomyces cerevisiae, copper green pseudomonas, staphylococcus aureus, Escherichia coli and Bacillus subtilis respectively, are mixed uniformly, incubated at room temperature for 2h, and photographed under the irradiation of an ultraviolet lamp. The operation was repeated 5 times.
(5) And (3) testing the fluorescence spectrum in each hole by using a microplate reader to obtain the response intensity of the fluorescent molecule sensing array to each microbial lysate.
(6) The data were analyzed and charted by principal component analysis (Canoco 4.5 software).
The fluorescence photograph of the fluorescence sensing array in this example after adding six kinds of microbial lysates is shown in fig. 2. As can be seen from FIG. 2, the fluorescence sensor array responds differently to each microbial lysate, primarily with a different intensity of the fluorescent molecular signal.
The signal of the fluorescence signal response intensity of the six microorganisms in this example is shown in FIG. 3. As can be seen in FIG. 3, each microbial lysate stimulates an array of fluorescent molecules to produce a specific array of fluorescence intensities.
The fluorescence signal data processing in this example is shown in fig. 4, and it can be seen from fig. 4 that six kinds of microorganisms are well distinguished after the principal component analysis processing.
Example 3
The fluorescence sensing array and the method for typing and identifying the microorganisms in the embodiment specifically comprise the following steps:
(1) preparing a solution of AIE molecules; respectively a1 (water soluble, 1mM), a2 (water soluble, 10mM), A3(DMSO soluble, 1mM), a4(DMSO soluble, 1mM), a5(DMSO soluble, 10mM), a6(DMSO soluble, 1mM), a7(DMSO soluble, 1 mM);
(2) and respectively adding 1 mu L of AIE molecular solution into test tubes, wherein the test tubes are numbered from 1 to 7, adding 5 mu L of graphene solution (the concentration is 0.4mg/ml) into tubes from 1 to 6, adding 0.5 mu L of graphene solution (the concentration is 0.2mg/ml) into tubes from 7, and uniformly mixing to prepare the fluorescent molecular sensing array. Two such sensor arrays were prepared in parallel.
(3) And additionally, preparing a set of fluorescent molecular sensing array without graphene oxide, which comprises the following specific steps:
(a) preparing a solution of AIE molecules; respectively a1 (water soluble, 1mM), a2 (water soluble, 10mM), A3(DMSO soluble, 1mM), a4(DMSO soluble, 1mM), a5(DMSO soluble, 10mM), a6(DMSO soluble, 1mM), a7(DMSO soluble, 1 mM);
(b) adding 1 mu L of AIE molecule (A1-A7) solution into seven test tubes, wherein the test tubes are numbered from 1 to 7, adding 5 mu L of aqueous solution into the tubes from 1 to 6, adding 0.5 mu L of aqueous solution into the tubes from 7, and uniformly mixing to prepare the graphene-free fluorescent molecule sensing array. Two such sensor arrays were prepared in parallel.
(c) And respectively adding bacterial lysis solutions (pseudomonas aeruginosa and escherichia coli) into the two sets of fluorescent molecular sensing arrays, incubating for 2 hours, and photographing under the irradiation of an ultraviolet lamp.
The fluorescence photograph data in this example is shown in fig. 5, a, the interaction between the bacterial lysate of pseudomonas aeruginosa and escherichia coli and the AIE molecule not containing graphene oxide is shown in the upper part of the fluorescence photograph, and the relative fluorescence intensity images generated by the two bacterial lysates are shown in the lower part of the fluorescence photograph. And b, the two bacterial lysates and the fluorescent molecular sensing array added with the graphene oxide interact with each other, the upper part is a fluorescent photograph picture, and the lower part is a relative fluorescence intensity picture generated by the two bacterial lysates. As can be seen from fig. 5, the fluorescent molecular sensing array without graphene oxide responds to both of the two bacterial lysates, and the two bacteria cannot be distinguished due to the same degree of response. And the two bacterial lysates can be well distinguished by adding the fluorescent molecular array of the graphene oxide. The reason for this is the influence of the competitive effect induced by graphene oxide on the promotion of the selectivity of the sensing array. A schematic diagram of the principle of the graphene oxide-induced competitive effect is shown in fig. 6.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
Claims (4)
1. A method for typing and identifying microorganisms by a fluorescence sensing array is characterized by comprising the following steps:
uniformly mixing the fluorescence sensing array and the microbial lysate for incubation, and performing typing identification on the microbes according to the difference of generated fluorescence signals;
the microorganisms comprise candida albicans, saccharomyces cerevisiae, copper green pseudomonas, staphylococcus aureus, escherichia coli and bacillus subtilis;
the fluorescence sensing array is formed by mixing an aggregation-induced emission molecular solution with the structural formula of A1-A7 and a graphene oxide solution; the concentration of the aggregation-induced emission molecule solution is 1-10 mM, the concentration of the graphene oxide solution is 0.2-0.4 mg/ml, and the volume ratio of the aggregation-induced emission molecule solution to the graphene oxide solution is 1 (0.5-5);
2. the method for typing and identifying a microorganism according to claim 1, wherein: the incubation time was 2 h.
3. The method for typing and identifying a microorganism according to claim 1, wherein: the generated fluorescence signal is collected by a microplate reader or a fluorescence spectrometer, and the collected signal realizes the typing identification of the microorganism by a principal component analysis method.
4. A microorganism typing identification kit used in the method for typing identification of microorganisms by the fluorescence sensor array according to claim 1, characterized in that:
the microorganism typing and identifying kit comprises a fluorescence sensing array, wherein the fluorescence sensing array is formed by mixing an aggregation-induced emission molecule solution with the structural formula of A1-A7 and a graphene oxide solution; the concentration of the aggregation-induced emission molecule solution is 1-10 mM, the concentration of the graphene oxide solution is 0.2-0.4 mg/ml, and the volume ratio of the aggregation-induced emission molecule solution to the graphene oxide solution is 1 (0.5-5);
the microorganism comprises Candida albicans, Saccharomyces cerevisiae, Pseudomonas aeruginosa, Staphylococcus aureus, Escherichia coli and Bacillus subtilis.
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